Project summary The goal of this proposal is to reveal how certain transcription factors can overcome different forms of silent chromatin to activate new genetic networks and elicit cell fate changes, ultimately to reprogram cells at will. Work on this grant led to the discovery that among transcription factors that specify new cell fates, a subset has the ability to bind their DNA target motif on nucleosomes, in the context of silent chromatin. We have termed these nucleosome-binding transcription factors pioneers because they can open up silent and previously unmarked chromatin, making it accessible to other factors. The insight has illuminated development, cell reprogramming, circadian rhythms, and transcriptional deregulation in cancer. By comparing various fate-changing transcription factors, we discovered that the ability to bind nucleosomes can be inferred by the presence of a short alpha-helix for sequence recognition in the DNA binding domain (DBD). Yet we find non-DBD protein domains are necessary to make an underlying nucleosome accessible, representing an open state of chromatin, and non-DBD domains of different pioneer factors elicit different patterns of accessibility. Few studies focus on how pioneer factors engage chromatin and expose underlying nucleosomal DNA, and pioneers can vary with regard to their targeting silent chromatin in different stages of compaction. We found that FoxA pioneer factors have a conserved, non-DBD alpha- helical domain that interacts with core histones and is necessary for chromatin opening in vitro, apparently by displacing inter-nucleosome interactions within the chromatin fiber. The FoxA alpha-helical domain is essential for opening chromatin in early endoderm development and for embryonic viability. Single- molecule imaging of HALO-tagged core histone, FoxA, and other transcription factors revealed that nucleosome binding confers the ability of pioneer factors to exist in the lowest mobility domains in chromatin, consistent with pioneering activity. It remains to be determined, and we will reveal, how core histone-pioneer factor interactions enable silent chromatin engagement, elicit different patterns of local chromatin opening, and thereby enable new gene networks and cell fate changes. We will use our findings to modulate pioneer factor structures and heterochromatin to enhance cell fate changes, testing cell products in vivo. Aim 1. Determine how pioneer factors interact with core histones in nucleosomes, and how such interactions lead to targeting and different patterns of accessibility in compacted chromatin in vitro. Aim 2. Determine the basis for different pioneer factors' selective access and opening of different forms of silent chromatin in vivo, and use the information to enhance cell reprogramming. Our proposal presents a new thesis: that optimizing pioneer factors while selectively disassembling heterochromatin provides an optimal way to generate new cell fates that are useful for biomedical purposes.